2004
DOI: 10.1128/aem.70.2.943-953.2004
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Development of Additional Selectable Markers for the Halophilic ArchaeonHaloferax volcaniiBased on theleuBandtrpAGenes

Abstract: Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these gene… Show more

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Cited by 391 publications
(672 citation statements)
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“…These large differences presumably explain why no recombinant expression of the alcohol dehydrogenase ADH/D1 was observed in E. coli. The engineered H. volcanii expression system (Allers 2010 ;Allers et al 2004Allers et al , 2010 was capable of producing moderate amounts of protein in shaker-flask cultures, the essential step for the biotechnological use of halophilic enzymes remains a large-scale production in bioreactors. The method established in this study facilitates straightforward batch cultivation of H. volcanii H1895 in a stirred-tank bioreactor using optimised settings and media.…”
Section: Discussionmentioning
confidence: 99%
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“…These large differences presumably explain why no recombinant expression of the alcohol dehydrogenase ADH/D1 was observed in E. coli. The engineered H. volcanii expression system (Allers 2010 ;Allers et al 2004Allers et al , 2010 was capable of producing moderate amounts of protein in shaker-flask cultures, the essential step for the biotechnological use of halophilic enzymes remains a large-scale production in bioreactors. The method established in this study facilitates straightforward batch cultivation of H. volcanii H1895 in a stirred-tank bioreactor using optimised settings and media.…”
Section: Discussionmentioning
confidence: 99%
“…The culture was incubated at 37 °C at 170 rpm. To the culture, 1 mM isopropyl-d-thiogalactopyranosid (IPTG) was added to induce recombinant 600 55.5 g L CaCl ) in a 250-mL shaker-flask (Allers et al 2004 ). This pre-culture was used to inoculate 1 L of sterile Hv-YPC medium and incubated at 45 °C at 170 rpm.…”
Section: Growth In Shaker-flasksmentioning
confidence: 99%
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“…This has enabled the establishment of gene knock-out systems in Halobacterium 77,78 , Hfx. volcanii 79,80 , and T. kodakaraensis 53 (see Table 2). The salient features of these systems are that the uracil marker can be reused (see Figure 1b), and that transformation is carried out using circular DNA (transformation using linear DNA is inefficient in some species).…”
Section: Transformationmentioning
confidence: 99%
“…Unless the mutant has a readily screened phenotype, the deletion must be verified by Southern blotting. c | Variant of the pop-in/pop-out method for gene deletion, where the gene is replaced with a marker allowing direct selection 80 . d | Combination of gene replacement (with ura marker) and pop-in/pop-out method, suitable for generating point mutants 77 .…”
Section: References Notesmentioning
confidence: 99%